MINI-REVIEW

The medicinal Agaricus mushroom cultivated in Brazil:biology, cultivation and non-medicinal valorisation

Michèle L. Largeteau &

Régulo Carlos Llarena-Hernández &

Catherine Regnault-Roger & Jean-Michel Savoie

Received: 30 June 2011 /Revised: 19 September 2011 /Accepted: 2 October 2011 /Published online: 18 October 2011# Springer-Verlag 2011

Abstract Sun mushroom is a cultivated mushroom exten-sively studied for its medicinal properties for several yearsand literature abounds on the topic. Besides, agronomicalaspects were investigated in Brazil, the country themushroom comes from, and some studies focus on thebiology of the fungus. This review aimed to present anoverview of the non-medicinal knowledge on the mush-room. Areas of commercial production and marketingtrends are presented. Its specific fragrance, taste, nutritionalvalue and potential use of extracts as food additives arecompared to those of the most cultivated fungi andlaboratory models. The interest of the mushroom forlignocellulosic enzyme production and source of biomole-cules for the control of plant pathogens are shown.Investigation of genetic variability among cultivars isreported. Growing and storage of mycelium, as well ascultivation conditions (substrate and casing generally basedon local products; indoor and outdoor cultivation; diseasesand disorders) are described and compared to knowledge onAgaricus bisporus.

Keywords Agaricus blazei . Agaricus brasiliensis .

Agaricus subrufescens . Biology . Cultivation . Geneticvariability . Marketing . Non-medicinal use . Pathology .

Production areas

Introduction

Gourmet mushrooms may contribute to the development ofa new agriculture by addressing the consumer demand andsome of the non-nutritional uses of agricultural productions.Mushrooms have been consumed by humans since ancienttimes, not only as a part of the normal diet but also as adelicacy due to their desirable taste and aroma, and asnutraceuticals. Important cultivated edible and medicinalmushrooms belong to the Agaricus genera and aresaprophytic fungi acting as secondary decomposers inforest litters. Once the value of recycling with gourmet andmedicinal mushrooms is clearly understood, wastes can beviewed as positive products, at least in terms of providing neweconomic opportunities and positive environmental conse-quences. Among secondary decomposers, an Agaricusspecies is today widely used and studied for its medicinaland/or therapeutic properties. Kawagishi et al. (1989) werethe first to separate an active anticancer compound from itsfruiting bodies. Since that time, numerous works wereperformed on the medicinal properties of the mushroom.Several reviews gave an overall view on the topic (Wasser2002; Firenzuoli et al. 2008; Oliveira Lima et al. 2011).

According to most of the published articles, themushroom cultivated in Brazil is native to the São PauloState, Brazil. It was formerly known in literature asAgaricus blazei Murrill (sensus Heinemann). In the lastfew years, several articles focused on the clarification of itsbotanical name (Kerrigan 2005, 2007; Wasser et al. 2002,2005) and agreed that the former name was wrong. Bystudying other specimens in addition to the cultivatedstrains, two names have been proposed Agaricus subrufes-cens Peck or Agaricus brasiliensis Wasser et al. Currently,many publications refer to A. brasiliensis for the cultivatedmedicinal mushroom originating from Brazil.

M. L. Largeteau (*) :R. C. Llarena-Hernández : J.-M. SavoieINRA, UR1264, MycSA,33883 Villenave d’Ornon, Francee-mail: largeteau@bordeaux.inra.fr

C. Regnault-RogerUPPA, UFR Sciences et Techniques,64012 Pau Université Cedex, France

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Various vernacular names have been given to thiscultivated medicinal mushroom. It is popularly knownin Brazil as Piedade mushroom, medicinal mushroom(cogumelo medicinal), Sun mushroom (cogumelo dosol), God's mushroom (cogumelo de Deus), and, due to itsfragrance and taste, as the Almond Portobello (AlmondMushroom; Portobello de amêndoa) (Colauto et al. 2002;Camelini et al. 2005; Dias et al. 2004; Gene 2009;Kopytowski Filho et al. 2006). The mushroom is calledHimematsutake, Agarikusutake and Kawariharatake inJapan, and Ji Song Rong in China (Firenzuoli et al. 2008).

A. brasiliensis has reached the uppermost ranks amongthe best of all gourmet and medicinal mushrooms. There isan extensive literature on this mushroom in which foodsciences, biotechnology, pharmacology and medicine arethe main topics whilst agronomy, marketing and non-medicinal use are far less represented. This review dealswith the biological characteristics of the Brazilian medicinalmushroom, its cultivation and non-medicinal valorisation.

Areas of commercial production and marketing

The medicinal mushroom A. brasiliensis was imported intoJapan from Brazil in 1965. Since 1988, it is cultivated on acontract basis in various parts of Japan and in Indonesia(Mizuno et al. 1990). In Brazil, it is produced at acommercial scale since the early 1990s (Braga et al.2006), in regions close to the Atlantic coast, of moderateto warm temperatures and of high humidity, from Septemberto April. The production areas have expanded from São Paulostate (still the most important producing region) to other statessuch as Minas Gerais (Southeast), Paraná, Santa Catarina andRio Grande do Sul (South), Bahia and Ceará (Northeast)(Mendonça et al. 2005; Neves et al. 2005).

Japan has become the greatest importer of A. brasiliensisproduced in Brazil (Dias et al. 2004). Indeed, during 2001,approximately 37 tons of dehydrated mushrooms wereexported from Brazil, principally to Japan (Eira 2003).Exportations sharply increased from 1996 to 1998 (from 7to 30 tons a year), were steady until 2001 (33 tons), thendecreased in 2002 and 2003 down to 20 tons, due tocompetition with other producers and the economic crisis inJapan (Mendonça et al. 2005). Decline in Japaneseimportations occurred for three consecutive years (2006–2008) and the market suffered a loss of 76% because ofproblems found in mushrooms imported from othercountries (SECOM 2010). Although the Brazilian productwas considered of higher quality, this did not prevent adecrease in Japanese consumption. In 2009, the marketstabilized, with a slight increase in exportations, but it is notpossible to predict whether the market will return to thelevels of 2004 (Dias 2010). Brazilian growers export to

several countries including Australia, Bolivia, Germany,Italy, Spain, South Africa, Thailand, the USA, India andKorea. At the same time as exportations have decreased,there has been a noticed improvement of the local marketdue to the interest of the Brazilian population for themushroom benefits (Mendonça et al. 2005).

Mushrooms are harvested when they reached the highestbiomass, which is the button stage with intact veilmembrane enclosing the gills (Mendonça et al. 2005). Thisstage reaches the highest market value for exportation(Camelini et al. 2005). The mushroom is traditionally soldin dehydrated form, as it is mainly consumed as nutraceu-tical, after being ground to powder. The dehydrationprocess includes washing and brushing to eliminate cappigment, followed by anti-oxidant treatment (ascorbic acid,3–5 gl−1 water) and drying. According to market standards,dried mushrooms are classified as grade Extra, A, B or C,following criteria of colour (straw colour, pale yellow),morphology (stalk aspect) and size (Mendonça et al. 2005;Minhoni et al. 2005).

In 2006–2007, the annual production in Brazil reached40 tons of dehydrated mushrooms (Tomizawa et al. 2007).Sliced dehydrated mushrooms, paid R$60–180 per kilo-gramme to the producer, were commercialised by whole-salers, mainly to Japan (Eira 2003). Mushroom price isvariable, depending on the region. Values ranged from US$28.6 to 71.4 per kilogramme of dried mushrooms, withaverage price of 50 US$ for grade A mushrooms(Mendonça et al. 2005). In São Paulo State, during 2004–2005, the average value for dried mushrooms was R$180–200 for grade Extra, R$150–180 for grade A, R$70–120 forgrade B and R$30–50 for grade C (Minhoni et al. 2005).Commercialization prices, low investment costs and fastreturn of invested capital make cultivation financiallyattractive. In Brazil, in social terms, it creates jobs, settingfamilies down to the land, as it demands a small cultivationarea. It is adapted to family agriculture, based on smallfarms (Cavalcante et al. 2008).

Apart from the large development in Brazil, A. brasiliensishas been produced at large scale in China since 1990s (Wanget al. 2010), whilst in Korea, cultivation started in 1996(Mizuno 2000). Nowadays, the mushroom is cultivated atthe industrial level in Brazil, Japan, China, Taiwan andKorea (Gregori et al. 2008). In 2001, Takadu et al. (2001)estimated that the production in Japan reached approximately100–300 tons of dried fruiting bodies every year. China,Japan and Korea are important competitors towards Brazilfor commercial production (Braga et al. 2006).

A. brasiliensis benefits from soil microflora and warmtemperature, making it an ideal candidate for outdoorcultivation in the subtropics. Due to the temperature neededfor fruiting, it might be an alternative to Agaricus bisporuscultivation during the warm season in Europe, for reducing

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cropping energetic cost. The almond taste might put a curbon its commercialisation as a gourmet mushroom, consum-ers being used to A. bisporus taste, but commercializationas nutraceutical can be expected because of the increasingdemand in natural products improving human health andwelfare.

Fragrance, taste and nutritional value

As indicated by one of its vernacular names, A. brasiliensisis characterised by a pleasant almond flavour. Benzalde-hyde and its precursor benzoic acid are the majorcomponents of the volatile fraction; the other contributorsto the flavour are benzyl alcohol, methyl benzoate and 4-hydroxybenzaldehyde. Benzoic acid (1,280–3,100 mg/kgdry weight), may contribute to the excellent shelf life of themushroom and presumably, by conversion to benzaldehydeand benzyl alcohol, to the development of the almondflavour when reconstituting the commercial dried mush-room. Benzoic acid occurs also in significant amounts inseveral close relatives of A. brasiliensis suggesting that thiscompound could be a taxonomic marker (Stijve et al.2003).

Analyses of the non-volatile taste components in threespecies of dried mushrooms commercially available inTaiwan showed highest contents of monosodium glutamate-like (MSG-like), sweet and bitter taste components in A.brasiliensis (4.40, 2.62 and 4.73 mg/g dry weight (DW),respectively) compared to Agrocybe cylindracea andBoletus edulis. The bitterness in A. brasiliensis couldprobably be masked by the sweetness, mainly from thehigh amount of soluble sugars and polyols (Tsai et al.2008). Mannitol concentrations reached up to 22% onmushroom dry weight. Compared to the fruiting body, themycelium exhibited no almond flavour and was found poorin flavour components, mannitol and MSG-like amino acids(Stijve et al. 2003). In another study, dried mycelia of A.brasiliensis showed lower content of sweet components andhigher content of bitter components than Antrodia cam-phorata and Cordyceps militaris mycelia (Chang et al.2001). Contents of flavour 5′-nucleotides (5′-GMP, 5′-IMPand 5′-XMP), which also gave the umami (meaty) orpalatable taste, were higher in dried A. brasiliensissporophores (5.15 mg/g) than in A. cylindracea and B.edulis (2.44 and 2.01 mg/g, respectively) (Tsai et al. 2008)and comparable to the 4.19–6.13 mg/g reported for A.bisporus by Tseng and Mau (1999). Considering the threeranges of flavour 5′-nucleotides defined by Yang et al. (2001)(low, <1 mg/g; middle, 1–5 mg/g and high, > 5 mg/g), thecontents in A. brasiliensis and A. bisporus were in the highrange. Using the equation derived from sensory evaluation(Yamagushi et al. 1971), the Equivalent Umami Content

(EUC) value was high (135.90 g/100 g). The sensoryEUC value of A. brasiliensis might be beneficial for itsuse as foods or food flavouring materials or in theformulation of nutraceuticals and functional foods with apalatable umami taste (Tsai et al. 2008).The content offlavour 5′-nucleotides was far lower in the mycelium of A.brasiliensis (7.0 mg/g) compared to those found in A.camphorata and C. militaris mycelia (38.0 and 29.2 mg/g) (Chang et al. 2001). Therefore, the balance betweenMSG-like and sweet components would be responsiblefor the specific taste of the Brazilian medicinal mushroom(Tsai et al. 2008).

Besides its medicinal interest, A. brasiliensis is a food ofhigh nutritional value, rich in protein, fibre, minerals, withlow lipid content. The protein content of 32.8–35.9% (veilclosed) and 28.9–39.2% (veil open) measured by Eira(2003) in dried sporophores cultivated in Brazil (fourstrains) exceeded percentages reported for Volvarielladiplasia (28.5%), A. bisporus (26.3%), Pleurotus spp.(18.7–23.3%) and Lentinula edodes (17.5–19.5%). Con-versely, less fibre contents were found in A. brasiliensis(5.56–9.7%, veil closed; 6.9–11.8%, veil open) comparedto V. diplasia (17.4%), A. bisporus (10.4%), Pleurotus spp.(10.4–15.6%) and L. edodes (8.0–12.4%). Other worksperformed on the Brazilian medicinal mushroom revealedsimilar levels of protein (30–45%) and crude fibre contents(6–14%), and showed carbohydrate contents of 38–45%and lipid contents of 1–5%. Fruiting bodies contained highamounts of potassium (2.34–2.97%), phosphorus (0.75–0.9%) and calcium (0.04–0.07%) (Mizuno et al. 1990;Oliveira et al. 1999; Eira 2003; Shibata and Demiate 2003).Compared to A. bisporus strain used as control, A.brasiliensis cultivars had lower K, P, Se and Na, but higherBo, Cd, Cu, Sr and Zn contents, and the Co and V contentswere below the limit of detection. There were no differ-ences for Ba, Ca, Mg, Mn, Ni and Ti. Content in vitaminB1 was comparable in the Brazilian cultivars (0.48 mg/100 g DW) and L. edodes (0.40 mg) whilst that in A.bisporus was two times higher (1.14 mg). Similar contentsin Niacin were found in A. brasiliensis (40.9 mg/100 gDW) and A. bisporus (36.2 mg) whilst only 11.9 mg werepresent in L. edodes. Vitamin B2 content ranged between A.brasiliensis (0.90 mg/100 g DW), L. edodes (0.90 mg) andA. bisporus (4.95 mg) (Eira 2003). Mushrooms cultivatedin Japan showed similar content in vitamin B1 and niacin(0.3 mg and 49 mg/100 g DW) (Mizuno et al. 1990).Interestingly, in a recent study, A. brasiliensis contained farless agaritine (22–57 μg/g DW), a known carcinogen, thanA. bisporus (341±32 μg/g DW) (Koge et al. 2011). Despiteits low content in fibre, A. brasiliensis is an attractive foodproduct characterised by vitamin content in the same range,protein content higher than those present in the other fungiand the most cultivated in the world.

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As a consequence of the chemical composition, besidesthe medicinal effects, literature reports some investigationson commercial or potential use of mushroom extracts asfood additives. Aqueous extract of A. brasiliensis have beenapproved in Japan as food additive to provide an agreeablebitter taste (Kuroiwa et al. 2005). Da Silva et al. (2009)identified a potential use of methanol/water extract of themushroom as natural anti-oxidant in vegetal oil.

Production of lignocellulose degrading enzymes

Fungal laccases and manganese peroxidases serve severalpurposes in the industry. There is an increasing interest onthe use of cellulases and hemicellulases for the valorisationof the plant biomass. Lignocellulases are produced in thecultivation substrates throughout the mushroom crop, butproduction is differently regulated depending on thedevelopment stage of the mushroom. This has beenextensively studied for A. bisporus (Savoie 1998) but notfor A. brasiliensis. Some works focused on comparisonsbetween fungal species for the production of enzymes bythe vegetative mycelium during several days followinginoculation. Laccases produced by A. brasiliensis, A.bisporus, Stropharia rugosoannulata and Volvariellavolvacea are similar; they are blue copper proteins with amolecular mass around 65 kDa and acidic pI between 3 and4 (66 kDa and pI 4.0 for A. brasiliensis laccase). TheBrazilian medicinal mushroom produced high amounts oflaccase during growth on complex liquid medium based ontomato juice (TJM), but only traces of the enzyme onsynthetic sugar-containing medium commonly used for theproduction of ligninolytic enzymes. Purified laccase wasstable at 20°C, pH 7.0 and 3.0, but rapidly lost its activity at40°C or pH 10. Whilst A. bisporus secretes manganeseperoxydase along with laccase in solid-state cultures oncompost, no manganese peroxidase was detected duringgrowth of A. brasiliensis in TJM. (Ullrich et al. 2005).

A. brasiliensis CS1 and Pleurotus ostreatus H1 provedto be efficient producers of holocellulases when grown onsolid-state medium supplemented with various proportionsof agro-industrial wastes (banana stem, sugarcane bagasse,cotton, corn and soybean residues). Enzyme inductiondepended on the nature of the substrate and the mushroomspecies. Cotton residue (10%) was the best carbon sourcefor xylanase, mannanase, endoglucanase and FPAse activ-ities (1,349, 206, 315 and 180 IU/l, respectively) in crudeextracts, whilst polygalacturonase activity (451 IU/l) wasobserved with 5% sugarcane bagasse, the most bulkmaterial used in mushroom compost in Brazil. Both fungireleased the highest quantities of reducing sugar after 72 hwith sugarcane bagasse (10%) and produced the highestamount of protein with banana stems (10%), but higher

values were obtained with CS1 (11.6 mg and 344 μg permilliliter of crude extract) compared to P. ostreatus(2.33 mg/l and 184 μg/ml) (Siqueira et al. 2010).

Industrial production of lignocellulolytic enzymes by A.brasiliensis in solid-state fermentation or by extractionfrom spent cultivation substrates is a new opportunity ofuse of this mushroom that is worthwhile to study.

Use in plant protection and other valorisation

Besides the well-known medicinal effects, A. brasiliensisextracts showed potential application in organic agriculturefor the control of plant pathogens. Several works reportedefficiency against various plant pathogens. Di Piero et al.(2010) compared the efficiency of aqueous extract from L.edodes and A. brasiliensis to protect passion flower(Passiflora edulis) and Chenopodium quinoa againstCowpea aphid-borne mosaic virus (CABMV). Pre-treatment of passion flower leaves with A. brasiliensisextracts before inoculation with CABMV reduced virusinfection by 66% (extract from ABL 99/29) and by 80–100% (extracts from ABL 99/28 and ABL 99/26). Three L.edodes strains gave less or no efficient extracts. Nosignificant systemic protection was observed whatever theextract tested. No protection of the extract from ABL 99/26was observed in experiments involving CABMV transmis-sion by aphid vectors. Other investigations, performed withisolates Abl-11 and Abl-28, showed potential to induceresistance in eggplant against bacterial wilt caused by thebacterium Ralstonia solanacearum. The mushroom aque-ous extracts caused significant reduction in the occurrenceof wilted leaves, when applied 2 days before inoculation.No protection was observed with aqueous extracts from twoL. edodes isolates (Silva et al. 2008). Crude extracts fromthe isolate ABL 29/99 (before and after veil opening)showed inhibitory effect against spore germination of theTriticum aestivum pathogen Puccinia recondita, but wereless efficient than extracts obtained from L. edodes LE95/01 (Fiori-Tutida et al. 2007). Aqueous extracts from A.brasiliensis and L. edodes sporophores reduced the forma-tion of new lesions caused by the ascomycete Guignardiacitricarpa, the causal agent of citrus black spots, in thesweet orange fruits (Citrus sinensis var. Valencia)(Pascholati et al. 2007). It is obvious from these inves-tigations that A. brasiliensis isolates exhibited variabilityin their biocontrol effect and, in some cases, proved to bemore efficient than L. edodes.

Besides, ethanolic extract obtained from compost afterA. brasiliensis crop appeared as a potential biotic elicitor ofresistance against Corynespora cassiicola in the cucumberplant (Cucumis sativus), showing a potential valorisation ofthe spent compost (Ueda et al. 2008). Spent compost also

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appears an interesting alternative to chemicals to promoteplant and animal growth. Fresh spent mushroom substrate(SMS) from A. brasiliensis production was a good sourceof N, P, and K and was an excellent supplement for lettucegrowth promotion. At 10% supplementation, lettuce aerialdry weight increased 2.2 and 1.3 times compared to thecontrol (without SMS) and NPK treatment. In contrast,supplementation with L. edodes SMS provided a drasticreduction in lettuce development. Therefore the use of A.brasiliensis SMS may be an excellent alternative to thechemical amendments when considering organic produc-tion of horticultural crops and Eucalyptus growing (pulpproduction) in artificial forests. SMS also showed potentialfor remediation of biocides possibly due to improvedmicrobial diversity and enzymatic activity (Ribas et al.2009). Substitution of antibiotics by A. brasiliensis SMSwas evaluated in chicken farming. Diet with 0.2% SMSprovided the highest weight gain and the best feedconversion (Machado et al. 2007).

Genetic variability

Several authors have investigated the genetic polymorphismamong cultivated strains of the Brazilianmedicinal mushroom.Neves et al. (2005) compared genetic variability among sixstrains provided by Brazilian spawn makers or isolated fromfruiting bodies collected in mushroom farms from differentStates (São Paulo, Minas Gerais, Santa Catarina and Paraná).RFLPs of the ITS region did not show polymorphism amongthe six strains. Genetic distances obtained from RAPDprofiles varied from 0% to 14%, and separated the strain22, originating from Santa Catarina, from the other strainswhich grouped together. Strain 22 also differed from theothers by its lowest in vitro mycelial growth rate and wasconsidered by mushroom growers to have low productivity.Colauto et al. (2002) performed RAPD on five strainsobtained from mushroom growers in São Paulo and RioGrande do Sul states, and found that three isolates (ABL97/11, ABL 99/25 and ABL 99/29) were probably clones,whilst ABL 99/28 and ABL 99/26 (re-isolated from Jun17-Japan) exhibited little genetic polymorphism. Strains 99/29and 99/26 showed distinct morphological differences despitemaintaining the characteristic pattern of the species. Later,using a far higher number of primers, Tomizawa et al. (2007)analysed nine Brazilian cultivars, including ABL 97/11, ABL99/25 and ABL 99/29. A group of six isolates (CS1, CS3,CS4=ABL97/11, CS6, CS8 and CS9) showed a high geneticsimilarity and were considered isolates of the same origin orclones. Isolates CS7=ABL99/29, CS5=ABL99/25 and CS2revealed 91.3%, 88.7% and 60.6% similarity with the group,respectively. Referring to PhD theses, the authors reportedthat cellulose degradation ability, cytological and physiolog-

ical aspects of CS2 also distinguished this isolate from theothers. RAPD performed by Marques et al. (2006) on threesets of cultivars, each from a different Brazilian producer,showed that only 2.7% of the 160 primers tested generatedpolymorphism but did not form separate groups. Smalldifferences in morphology were attributed to environmentalfactors during cultivation. Molecular analyses performed oneight strains (SA515–520, SA 555 and SA527) provided byJapanese growers and strain SA514 from a Brazilian growershowed clear genetic differences between the Braziliancultivar and the Japanese-cultivated strains; the latter groupedtogether (Fukuda et al. 2003; Mahmud et al. 2007). All theseworks revealed great homogeneity for commercial strainsanalysed either in Brazil or in Japan, but in each country, thestrains currently cultivated probably derived from a single ora very few sporophores. Local cultivation conditions andstrain improvement by laboratories or producers mightexplain differences found between Brazilian and Japanesecultivars despite the latter are of Brazilian origin.

Two needs were identified for improving the knowledgeof biodiversity in cultivated strains of the medicinalmushroom. First, it is necessary to develop efficientmarkers. Since transposons of the same type were foundin high number of copies in each genome of threebasidiomycetes, they appear to be excellent markers forstudying genetic structures of natural populations and forconstructing linkage maps, especially in Agaricus species(Sonnenberg et al. 2004). Other markers like ISSR (Guan etal. 2008) and especially SSR that proved highly efficient indifferentiating A. bisporus strains (Foulongne-Oriol et al.2009) might also be developed. High genetic homogeneitywas observed among cultivars, so there is a need toprospect for wild strains for trying to find genetic diversitysubsequently available for the improvement of the cultivatedstrains through breeding programmes. Besides, samplesof the Brazilian strains named A. braziliensis andformely A. blazei proved to be genetically similar to orvery close to, and inter-fertile with, the North Americanpopulation of A. subrufescens (Kerrigan 2005), showingthat there is a potential for the production of new hybrids(Kerrigan and Wach 2008). Specimens of divers geo-graphical origins belonging to this species complex werecollected and studied (Kerrigan 2005; Peter-Valence et al.2011) opening new opportunities for genetic improvementthrough the use of new sources of genetic variability.However, level of medicinal activities in hybrids must beanalysed.

In vitro mycelial growth and storage conditions

Mycelial growth in vitro is well documented, although mostof the publications refer to conditions adapted for the

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production of compounds of medicinal interest (exo-polysaccharides mainly), and show that conditions differfor the production of biomass and that of nutraceuticals(Fan et al. 2007; Gao and Gu 2007). Some information onthe method and optimization of culture medium to producesubstantial mycelial growth is available. Composition ofculture media, derived from those used to grow A. bisporus,and procedures showed little differences. Glucose andsaccharose were found the most effective carbon sourceand casamino acids as the most effective nitrogen sourcefor mycelial growth of A. brasiliensis on agar medium (Shuand Xu 2007). In bioreactor fermentation conditions,glucose yielded the highest biomass, followed by maltose,saccharose and fructose (Zou 2006). Consistent mycelialgrowth rates were observed on agar medium adjusted atpH 5.5 to 7.0 with HCl or NaOH, but strong inhibitionoccurred at pH 8.0 adjusted with NaOH or CaCO3 (Neveset al. 2005; Colauto et al. 2008).

Various optimal temperatures have been reported in theliterature. All but one of the six Brazilian cultivarscompared by Neves et al. (2005) showed optimal growthtemperature of 28°C or 30°C. More recently, Colauto et al.(2008) observed that five A. brasiliensis strains can developmycelium at temperatures between 22°C and 34°C, withoptimal temperature ranging from 30.2°C to 30.8°Cdepending on the strain.

The Brazilian medicinal mushroom is known to be killedby prolonged exposure to temperature of 4°C or lower.However, short-time preservation at this temperature ispossible. Zou (2005) stored mycelium grown on agarmedium for about 2 weeks at 4°C. Other authors main-tained mycelium cultures at higher temperature. Forinstance, Andrade et al. (2008) prepared agar culturessubmerged with sterilized mineral oil, and stored at 8°C,whilst Fan et al. (2007) maintained stock cultures on agarmedium at 20°C and sub-cultured every 3 months. In ourlaboratory, we currently store agar cultures at 12°C forseveral months.

For spawn making, mycelium is commonly inoculated tocereal grains (wheat, rice, titricum and sorghum) withadded gypsum and calcitic limestone, previously cookedand sterilized. Incubation lasts for 21–25 days, generally at28°C (Minhoni et al. 2005; Siqueira et al. 2009; Colauto etal. 2010). Commercial spawn is packaged in polyethylenebags. It can be stored at 10–12°C for no longer than2 months, and must be put out of the fridge 24 h before useto be at room temperature at spawning time. Spawnmaintained at 25–28°C should be used within a maximumperiod of 20 days (Minhoni et al. 2005).

However, the lower temperature suitable for long-time storage of mycelium cultures is still unknown, aswell as the lower temperature allowing mycelium todevelop.

Mushroom cultivation

In Brazil and Japan as well, A. brasiliensis is cultivated by aprocedure based on the method used to grow A. bisporus(Mahmud et al. 2007; Mantovani et al. 2007). Despite itseconomic importance, cultivation in Brazil is based uponlow-level technologies, and production level is very lowcompared to that of A. bisporus (Siqueira et al. 2009). A.brasiliensis and A. bisporus are native to different environ-ments and could respond differently to substrate and casingformulations. Therefore, several research projects havebeen developed with the aim to find the best rawingredients, composting processes and casing materials.

Compost composition and disinfection

Mushroom cultivation could participate to bioconversion ofagricultural wastes which are cheap and easily available.Brazilian producers of substrate choose regional abundantmaterial efficient for cultivating the medicinal mushroom.Various grasses (Brachiara sp., coast-cross (Cynodondactylon), etc.) and straws (wheat, rice, corn, barley),sugarcane (Saccharum officinarum) bagasse, sunflowerseed hulls, poultry and horse manure, nitrogen supplements(cotton seed, wheat and soybean bran) are commonly used,supplemented with urea, anmmonium sulphate, plaster andlimestone (Mendonça et al. 2005; Minhoni et al. 2005; Ziedand Minhoni 2009; Matute et al. 2010). Other by-productsare under investigation in several countries. In Brazil, largeamounts of cassava (Manihot esculenta) fibres resultingfrom industrialization process are used as animal feed, butalso burned or dumped on the soil, increasing environmen-tal damage. A mixture of soybean and cassava fibressustained consistent mycelial colonisation (Zaghi Junior etal. 2010) and might be tested for mushroom production.Recently, with the problem of the sugar cane burning forharvesting, research is being done to replace the sugar canebagasse by sugar cane straw for the production of thecompost (Zied, pers. com.). Chicken manure was appropriatefor fruiting body production (commercial strain 7700) whenmixed with wheat straw at a rate of 80:100 (Gregori et al.2008). In some regions in China, there is a need to selectlocal, cheap raw material as substrate for ABM production.Compost consisting in cotton seed hulls, rice hulls, cowmanure and wheat bran was proposed by Zhou et al. (2010).In Shanxi province, approximately 100,000 tons per year ofAsparagus officinalis L. is produced, generating largeamounts of straw. Substrate composed of asparagus straw,cotton seed hull, soybean cake (6:3:1 DW) suited for ABMSH26 cultivation (Wang et al. 2010).

The composting procedure to prepare substrate forcultivating A. brasiliensis (Colauto et al. 2010; Zied andMinhoni 2009; Zied et al. 2010) is similar to the two-phase

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short process defined by Laborde et al. (1993) for A.bisporus.

Spawn run

The composted substrate is commonly inoculated with 1–2% spawn (fresh weight) and incubated at 23–28°C for 15–30 days, depending on the strain and temperature (Braga etal. 2006; Cavalcante et al. 2008; Minhoni et al. 2005;Siqueira et al. 2009; Wang et al. 2010). Besides, Gregori etal. (2008) observed that spawning at 2% and 5% ratesincreased the yield of strain 7700 by 26% and 29% inaverage, respectively, compared to yields obtained with 1%spawn in a cultivation substrate composed of wheat strawand chicken manure. From our observations, yield ofseveral Brazilian cultivars in A. bisporus commercialcompost depends on the mushroom strain more than onspawn percentage (1% vs 2%) (unpublished data).

Casing

In North America and Europe, Sphagnum peat moss issuccessfully used for A. bisporus cultivation. In Spain, fourA. brasiliensis isolates cultivated with five different casingmixtures based on soil and a Dutch commercial casing(peat) showed the higher yield with the Dutch commercialcasing (Zied, unpublished data). In Brazil, the commercialexploitation of peat is regulated by environment legislation,and good quality peat for mushroom production is noteasily found. Consequently, casing materials from localsoils (litholic, latosol, Rhodic Hapludox, Xanthic Haplu-dox, Humic Haplaquox, yellow–red argissoil) have beeneither traditionally used or tested for A. brasiliensiscultivation (Cavalcante et al. 2008; Siqueira et al. 2009;Zied and Minhoni 2009). Casing mixtures, based on soiland various compounds, such as washed river sand,vegetable charcoal, rice husk, coconut fibre, vermiculite,Eucalyptus sawdust, are disinfected at 60–65°C for 6–8 hbefore use (Minhoni et al. 2005; Colauto et al. 2010).Siqueira et al. (2009) demonstrated the importance of thechoice of the casing soil, regardless of the compostcomposition. Adding 30% of charcoal to subsoil improvesthe particle size distribution, decreases compacting, favoursgas exchanges and/or reduces the presence of potentialvolatile inhibitors, which is considered by some producersan improvement to the yield (Colauto et al. 2010).However, finding the best casing composition seemsdifficult. For instance, strain 04/49 yield was positivelycorrelated with silt amount and water-holding capacity, andnegatively correlated with clay amount and bulk density,but mushroom numbers and mushroom mean weightcorrelated differently with casing properties (Zied et al.2011). The quantity of casing material proved important for

mushroom yield, but depends greatly on cultivation con-ditions. Braga and Eira (1999) observed higher yields ingreenhouse with casing layers of 5–8 cm thick compared toa 3-cm layer, and no effect of casing layer thickness inbamboo-covered structures. Mendonça et al. (2005)reported casing depths ranging from 3 to 5 cm, dependingon the environmental variability of the growing house,whilst Minhoni et al. (2005) found that 5–8-cm casing layermaintained at 60–75% humidity gave the best productivity.

Fruiting induction and yield

Commercial cultivation takes place in climatic rooms andplastic green houses, under the process used for A. bisporusexcept the temperature. The plastic film covering the casinglayer during the 7–10 days of post-incubation is removedand primordium induction performed by increasing aera-tion, maintaining 85–90% RH and decreasing the temper-ature. Various temperatures were reported for fruitinginitiation: a slow cooling down from 28°C to 20°C(Kopytowski Filho et al. 2008), 20–22°C during 3 days(Minhoni et al. 2005), below 25°C (Mendonça et al. 2005),20°C (Colauto et al. 2010), 17±1°C (Gregori et al. 2008).After that, the culture were maintained at 22±1°C (Gregoriet al. 2008) and 23±2°C (Colauto et al. 2010).

In small-scale experiments, Horm and Ohga (2008)applied a photoperiod of 12-h light (500 lux)/12-h darkduring the fruiting period, but the mushroom developsperfectly in the dark.

Time to primordial onset is depending on the casingmaterials (Wang et al. 2008). The first flush occursapproximately 15–20 days after casing, depending on thestrain. Generally, the successive flushes are separated by15–20 days, but delay might be 30–60 days depending onthe environmental conditions. The cultivation cycle lasts90–120 days from casing. Longer cycles are not interestingfrom an economical point of view (Mendonça et al. 2005;Minhoni et al. 2005; Wang et al. 2008; Zied et al. 2010).

Compared to other cultivated Agaricus, the productivityof A. brasiliensis is low. Minhoni et al. (2005) reportedyields of 6.5% to 9.5% (FW). Zhou et al. (2010) reportedfresh mushroom yields of 9 kg/m2. Recently, Zied et al.(2011) estimated the average production in Brazil at 8–16%after 120 days of cultivation. Several works comparedmushroom yield under various cultivation conditions.Seasonal cultivation in Santa Catarina State is mostlyindoors, in plastic, wooden or brick houses, generallywithout equipment for climatic control (Mendonça et al.2005). Cultivation feasibility was evaluated in Ceará state,Brazil, by comparing the production in covered areas andoutdoor, in a bordering area of native forest. Thirtycentimetres of dried grass (Melinus minutiflora) were usedto protect the outdoor cultures. Cultivation in covered area

Appl Microbiol Biotechnol (2011) 92:897–907 903

leaded to only 2.7% yield, whilst outdoor (higher variationin maximal temperature and higher RH), cultures began tofruit earlier, and yielded 6.5%, which reached near theproductivity obtained in the traditional production areas(Cavalcante and Gomes 2005; Cavalcante et al. 2008).Braga et al. (2006) compared cultivation in rough structureof wood fenced by bamboo sticks, plastic green house andclimatic room (dark, 25±2°C, 80±5% RH, air renewal).The earliest mushroom initiation and shortest productioncycle occurred in the climatic room, but the highest yieldwas recorded in the plastic greenhouse, where the meandaily temperatures were higher compared to those recordedin the two other cultivation environments. The rusticstructure, with bamboo covering, was the least adapted forinitiation, yield and production cycle. The authors con-firmed their previous observation stating that the mushroomdevelops better under relatively high temperatures, close to30°C. In contrast, Eira (2003) recommended fallingenvironmental temperature when exceeding 28°C. Minhoniet al. (2005) observed higher productivity in plasticgreenhouse with temperature ranging from 15°C to 35°Cthan in cultivation room set at a constant temperature of25°C. But no primordium induction occurred under aconstant temperature of 15°C. Zied et al. (2010) obtainedhigher yield (15.5–16.1%) in plastic greenhouse comparedto controlled environment (12.4–12.9%) and consideredthat the weather during the experiment could explain thebetter result observed in greenhouse. These observationssuggest that daily temperature variations are more favour-able to A. brasiliensis mushroom yield than constanttemperature.

Biotic and abiotic disorders during cultivation

A. brasiliensis is a fungus of hot climate and lowtechnology systems. Temperature range (25–30°C through-out the production cycle) favours the emergence of pestsand diseases (Eira 2003; Nascimento and Eira 2007), andcontamination occurs frequently when adding supplementto the compost, either at spawning or before casing(Kopytowski Filho et al. 2008). Besides, frequentexchanges of compost and spawn among different Brazilianstates facilitated the rapid spread of diseases and pests(Mendonça et al. 2005). The major fungal competitorsobserved during cropping were those affecting A. bisporuscrops (Fletcher et al. 1989).

The use of pH around 7.0–7.5 for compost and casinglayer preparation would favour the establishment of A.brasiliensis while reducing fungal competition, especiallyTrichoderma spp. (green mould) that is usually presentunder tropical temperature and proliferating at pH 5–6. Buthigh percentage of composts contaminated by Trichoderma

sp. (green mould) was observed when the temperature ofthe compost was raised above 32°C after spawning(Kopytowski Filho et al. 2008). As observed in A. bisporuscultivation, Chaetomium olivaceum (olive green mould)develops when the temperature overcomes 62°C duringphase II of composting, leading to a decrease in thermo-philic microflora. The false truffle (Diehlomyces micro-sporus) is one of the main problems in the production of themedicinal mushroom in Brazil, mainly when the appropri-ate technology is not used. The disease was observed in2000 in the Brazilian states of São Paulo and Paraná, withcrop losses up to 90%. In Spain, the first report of D.microsporus occurred in 2010 (Gea et al. 2010). Whendeformed mushrooms, commonly called ‘pop corn’appeared, symptomless mushrooms no longer developed.D. microsporus needs high temperature (22–30°C) forascospore germination. The maximum growth speed hasbeen observed when the culture was incubated at 28–30°C(Eira 2003; Nascimento and Eira 2003, 2007). Species ofAspergillus, Emericelle and Penicillium genera have beenidentified from two kinds of compost (sugarcane bagasse/coastcross hay and cotton residue/coastcross hay). Asper-gillus fumigatus, a human pathogen, was also found in bothformulations, so preventive measures should be taken byworkers involved in compost production (Dias et al. 2009).Papulaspora sp. (brown plaster mould), known to developin wet compost has been observed (Minhoni et al. 2005).Coprinus sp., a competitor in compost, could developduring spawn run, revealing too high NH3 concentrationthat is unfavourable to mycelial growth. Coprinus and A.brasiliensis sporophores cohabit in infected mushroombeds, but infection after casing could result in deteriorationof the mushroom mycelium (Eira 2003). Other majorcompetitors observed in A. brasiliensis crops are Dactyliumdendroides (cobwed disease), Arthrobotrys sp. (brownmould), Stemonitis sp., Peziza sp. (cinnamon mould),Alternaria sp., Mucor sp. and Cladosporium sp. (Eira2003; Andrade et al. 2007). Competitors generally delayedthe time course for the flush and reduced the yield. In themajor cases analysed, contamination became more severeon the second or third year of cultivation, as observed for A.bisporus (Eira 2003).

There is a high incidence of pathogenic bacteria(Pseudomonas tolaasii) in Brazil conditions of cultivationdue the high air temperature and relative humidity.Bacterial rot associated to Mycogone or Lecanicillium(Verticillium) and damage caused by flies in too wet andinsufficient aeration conditions was reported. In Brazil, atthe beginning of the 2000s, dry bubble symptoms wereobserved in a single area of production, and were attributedto Lecanicillium fungicola (Eira 2003). Lately, some strainsare being selected and tested by presenting greaterresistance to Lecanicillium (Zied, personal communication).

904 Appl Microbiol Biotechnol (2011) 92:897–907

Pests andmites can affect crops. Dipters from three families,Sciaridae (Lycoriella spp.), Phoridae, especially Megaseliahalterata and Cecidomidae (Mycophila speyeri) are respon-sible for important qualitative and quantitative losses (Eira2003). The parasite Collembola entomobryidae was observedin mycelium and sporophore. Mites (Tarsonemus spp. andPymaephorus stercoricola) were detected in cultivationmaterials from various producers. They develop quickly atthe environment conditions suitable for A. brasiliensiscultivation: RH above 90%, and temperature 25–30°C.Nematodes identified as mycophagous (Aphelenchoidescomposticola and Ditylenchus myceliophages) or not(Rhabditus spp., Mesorhabditis spp. especially) causedamage in cultures. Most of these nematodes also infestA. bisporus crops in Europe, USA and Asia (Eira 2003).

Environmental conditions and chemicals could beresponsible for sporophore malformation. Poor aeration,excess of CO2, result in too thin and elongated stipes.Cracked caps appear with insufficient aeration and too lowRH (Eira 2003; Minhoni et al. 2005)

Conclusion

Literature shows valuable non-medicinal aspects of thecultivated strains of A. brasiliensis originating from Brazil,as its high nutritional value, its potential use for industrialproduction of lignocellulolytic enzymes and for applicationin organic agriculture for the control of plant pathogens andelicitation of resistance, and as an alternative to chemicalamendments. The published works on genetic variabilityconcern mostly cultivars. Mycological prospectings toenlarge collections of specimens should lead to advance inthe study of phenotypic and genetic diversity of the Agaricuscomplex (A. brasiliensis, A. subrufescens) and provide,through breeding experiments, high producing strains forwhich the medicinal activities will have to be evaluated.Indeed, the A. brasiliensis is mainly produced in Brazil andAsian countries on local substrates, with poor yieldscompared to A. bisporus. Improvement of cultivationconditions is needed. Several parameters (raw material,casing, temperature) must be studied to increase yield andadapt cultivation to temperate countries.

Acknowledgement This work was supported by a France–MexicoProject (project 115790 CONACYT, and ANR-09-BLAN-0391-01).RC Llarena Hernández would like to thank CONACYT, Mexico, forscholarship.

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